Chemical Functionalization of Plasmonic Surface Biosensors: A Tutorial Review on Issues, Strategies, and Costs

Abstract

In an ideal plasmonic surface sensor, the bioactive area, where analytes are recognized by specific biomolecules, is surrounded by an area that is generally composed of a different material. The latter, often the surface of the supporting chip, is generally hard to be selectively functionalized, with respect to the active area. As a result, cross talks between the active area and the surrounding one may occur. In designing a plasmonic sensor, various issues must be addressed: the specificity of analyte recognition, the orientation of the immobilized biomolecule that acts as the analyte receptor, and the selectivity of surface coverage. The objective of this tutorial review is to introduce the main rational tools required for a correct and complete approach to chemically functionalize plasmonic surface biosensors. After a short introduction, the review discusses, in detail, the most common strategies for achieving effective surface functionalization. The most important issues, such as the orientation of active molecules and spatial and chemical selectivity, are considered. A list of well-defined protocols is suggested for the most common practical situations. Importantly, for the reported protocols, we also present direct comparisons in term of costs, labor demand, and risk vs benefit balance. In addition, a survey of the most used characterization techniques necessary to validate the chemical protocols is reported.

Keywords: biomolecule pattern; characterization; functionalization; nanostructures; plasmonic sensors.

Figure 1

Pictorial description of the most important aspects of sensor functionalization.

Chart 1. General Structures of (a) an Antibody, (b) an Aptamer, and (c) a Sugar

Scheme 1. Oriented Immobilization of Ab Fragments on Gold Surfaces

Scheme 2. Coupling Strategies to SAM-Modified Au Surfaces Exploiting (a,b) Lysine Residues, (c,d) Cysteine Residues, or (e) Acid Residues on the Biomolecule

Scheme 3. Site-Specific Coupling of Biomolecules to Au Surfaces: (a) Coupling via Pro-Linker B Linker, (b) Coupling via Diels–Alder Cycloaddition Strategy, and (c) Coupling via Streptavidin–Avidin Strategy Mediated by CS₂

Scheme 4. Bioaffinity Coupling Using (a) Streptavidin–Biotin, (b) Protein A/Protein G Mediated via Classical NHS/EDC Chemistry, (c) CS₂ Chemistry, or (d) Polycysteine Tag Engineering

Scheme 5. (a and c) Chemical and (b) Bioaffinity Interaction Strategies To Achieve Orthogonal Functionalization in Nanostructured Biosensors

Figure 2

(A) Optical image of a water drop on a superhydrophobic surface. (B) Pictorial description of drop drying on a hydrophobic surface, delivering fluorescein molecules in silver-clustered hydrophilic domains. (C) Scanning electron microscopy (SEM) image showing the geometrical arrangements of Ag small-island films selectively grown inside wells surrounded by superhydrophobic domains. (D) Confocal fluorescence microscopy image in the dye spectral range of the pattern shown in panel (C). (Adapted with permission from ref (114). Copyright 2014, Wiley, Weinheim, Germany.)

Figure 3

(a) Gold pattern of the sensor chip on the PTFE film. Best experimental configuration: 40 μm pitch with duty cycle = 0.5, with a static contact angle of ∼100°. (b) Schematic diagram of functionalization of the sensor. (c) Fluorescence confocal microscopy image of the gold-patterned sensor.

Figure 4

Overall time and cost provisions for typical functionalization strategies.